Our long term interest is in identifying the characterizing genes involved in poxvirus pathogenesis. We propose to focus on three gene products of rabbitpox virus (RPV): ps/hr (B5R ORF), SPI, and SPI-2. These virulence genes reside in the terminal regions of the viral genome, and have homology either to complement control proteins (ps/hr) or to serine proteinase inhibitors (SPI-1 and -2). Wild type RPV is highly virulent, grows on a wide range of cell lines in vitro, and gives red, hemorrhagic pocks on the chorioallantoic membrane (CAM) of embryonated chicken eggs. Mutants in all three genes are attenuated in anima model systems (ps/hr and SPI-2), or show host range restriction (ps/hr and SPI-1), or produce white pocks containing inflammatory cells on the CAM (ps/hr, SPI-1 and - 2). The ps/hr gene has homology both to factor H of the complement pathway and to factor XIII of the coagulation pathway, and encodes a 45 kDa glycoprotein found in extracellular enveloped virus (EEV) and a soluble 35 kDa form which is secreted by a process independent of virus maturation. We plan to construct viruses synthesizing either the secreted or membrane-associated form only in order to identify which form of the ps/hr protein is responsible for control of inflammation and which is needed for full host range. Site-directed mutagenesis will then be used to identify critical domains of the protein. Biochemical assays based on the homologies shown by the ps/hr protein will be used to determine how the ps/hr protein inhibits inflammation. The RPV SPI-1 and SPI-2 proteins also regulate inflammation in eggs, and will be assayed for inhibition of interleukin-1beta converting enzyme (ICE) based on the known activity of the cowpox virus (CPV) SPI-2 homolog. The RPV SPI-1 protein is required for plaquing on A549 cells, and lack of this gene product results in the induction of apoptosis. The mechanism of this host range restriction will be analyzed further. In order to explore possible functional overlap between the SPI-1 and SPI-2 genes, which are about 45% identical, an RPV mutant lacking both genes will be constructed and tested. The different effects of single SPI-gene disruptions in RPV and CPV will be examined by gene swapping. We plan to screen overexpressed RPV SPI-1 and -2 and swinepox serpin proteins for inhibition of defined serine proteinases in vitro, and for inhibition of serine proteinases involved in CTL- and NK- mediated cytolysis of target cells. Interactions with other proteins will be screened genetically by means of the yeast two-hybrid system, and biochemically by use of tagged serpins. The results of these assays will be linked to an extensive analysis of the behavior of knockout and site- directed RPV mutants in eggs, mice and rabbits.